It has been known that the Toll gene is required to control dorsoventral patterning during the embryonic development of Drosophila (Cell 52, 269-279, 1988; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996), and for antifungal immune responses in adult fly (Cell 86, 973-983, 1996). It has been clarified that the Toll is a type I transmembrane receptor with an extracellular domain containing leucine-rich repeat (LRR) and that its cytoplasmic domain shows high homology to that of a mammalian interleukin-1 receptor (IL-1R) (Nature 351, 355-356, 1991; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996; J. Leukoc. Biol. 63, 650-657, 1998).
Recently, mammalian homologs of Toll, designated as Toll-like receptors (TLRs), have been identified, and so far, six families including TLR2 and TLR4 have been reported (Nature 388, 394-397, 1997; Proc. Natl. Acad. Sci. USA 95, 588-593, 1998; Blood 91, 4020-4027, 1998; Gene 231, 59-65, 1999). It has been known that the TLR families, as in the case of the IL-1R mentioned above, recruit IL-1R-associated kinase (IRAK) through the adapter protein MyD88 and activate TRAF6, and then activate NF-êB in the downstream (J. Exp. Med. 187, 2097-2101, 1998; Mol. Cell 2, 253-258, 1998; Immunity 11, 115-122, 1999). Further, the role of the TLR families in mammals is also believed to participate in congenital immune recognition as pattern recognition receptors (PRRs), which recognize bacterial cell common structures (Cell 91, 295-298, 1997).
It has been reported that one of such pathogen-associated molecular patterns (PAMPs) to be recognized by the PRRs is a lipopolysaccharide (LPS), a major component of the outer membrane of Gram negative bacteria (Cell 91, 295-298, 1997), that the LPS stimulates host cells and makes them produce various proinflammatory cytokines including TNFá, IL-1, and IL-6 (Adv. Immunol. 28, 293-450, 1979; Annu. Rev. Immunol. 13, 437-457, 1995), and that the LPS captured by LPS-binding protein (LBP) is delivered to CD14 on the cell surface (Science 249, 1431-1433, 1990; Annu. Rev. Immunol. 13, 437-457, 1995). The inventors of the present invention have constructed TLR4 knockout and TLR2 knockout mice, and have reported that the TLR4 knockout mouse is unresponsive to LPS, a major component of the outer membrane of Gram negative bacteria mentioned above (J. Immunol. 162, 3749-3752, 1999) and that a macrophage of the TLR2 knockout mouse decreased the reactivity of the TLR2 knockout mouse to the cell wall of Gram positive bacteria and peptidoglycan, a component of the cell wall (Immunity, 11, 443-451, 1999).
On the other hand, Mycoplasma is the smallest microorganism that can self-propagate, and is biologically classified into bacteria. However, unlike other bacteria, Mycoplasma does not have a cell wall, and therefore, it shows polymorphology and is unresponsive to cell wall synthesis inhibitors such as penicillin and cephem. Though there are seven kinds of Mycoplasma which are often separated from human, only Mycoplasma pneumoniae shows apparent pathogenicity and is known to cause respiratory infections such as upper respiratory infection, bronchitis and pneumonia. Recently, the present inventors have revealed that a bacterial cell component such as a mycoplasma-derived lipoprotein/lipopeptide causes vital reaction via TLR2 and MyD88 signaling pathways (J. Immunol. 164, 554-557, 2000). However, a protein that specifically recognizes a mycoplasma-derived lipoprotein/lipopeptide has been unknown, and consequently, the molecular mechanism that a mycoplasma-derived lipoprotein/lipopeptide activates immune cells has not been elucidated sufficiently.
Though in vivo responses to bacterial cell components are expected to vary depending on the difference of expression levels of each TLR on the cell surface, the contribution of individual members of the TLR family to signaling by bacterial cell components' stimuli in vivo remains to be elucidated. In addition, though it is known that a water-insoluble lipoprotein/lipopeptide that is present on a biomembrane etc. activates immune cells, a protein that specifically recognizes a mycoplasma-derived lipoprotein/lipopeptide has been unknown. An object of the present invention is to provide a non-human animal model unresponsive to a mycoplasma-derived lipoprotein/lipopeptide, whose function of a gene that encodes a protein that specifically recognizes a mycoplasma-derived lipoprotein/lipopeptide is deficient on its chromosome, particularly a non-human animal whose function of the TLR6 gene is deficient on its chromosome, which is useful for elucidating the contribution of individual members of the TLR family to signaling by stimulation with a mycoplasma-derived lipoprotein/lipopeptide in vivo, especially the role of TLR6 in vivo, and a method for screening an inhibitor or a promoter for a response to a mycoplasma-derived lipoprotein/lipopeptide with the use of the non-human animal model.
As aforementioned, as to TLR family in mammals, which is involved in congenital immune recognition as a pattern recognition receptor which recognizes bacterial cell common structures, six members of them (TLR1-6) have been reported (Nature 388, 394-397, 1997; Proc. Natl. Acad. Sci. USA, 95, 588-593, 1998; Gene 231, 59-65, 1999). However, a receptor that specifically recognizes a mycoplasma-derived lipoprotein/lipopeptide has been unknown. The present inventors have generated TLR6 konckout mice as follows: cDNA of TLR6 which had been identified was isolated from mouse gene library; a genetic site containing an intracellular domain and a transmembrane domain of the TLR6 gene was replaced with a neomycin-resistant gene, and a HSV-tk gene was introduced into each C-terminal side respectively, and ES cell clones doubly resistant to G418 and ganciclovir were screened; the ES cell clones were injected into blastocysts of C57BL/6 mice; TLR6 knockout mice whose function of TLR6 genes is deficient on their chromosomes were born through the germline at the expected Mendelian ratios. Subsequently the present inventors have found that TLR6 is a receptor protein that specifically recognizes a mycoplasma-derived lipoprotein/lipopeptide by comparing/analyzing the TLR6 knockout mice, wild-type mice and TLR2 knockout mice, and the present invention has completed.